Summary

The Janus kinase (Jak) family is one of ten recognized families of non-receptor tyrosine
kinases. Mammals have four members of this family, Jak1, Jak2, Jak3 and Tyrosine kinase
2 (Tyk2). Birds, fish and insects also have Jaks. Each protein has a kinase domain
and a catalytically inactive pseudo-kinase domain, and they each bind cytokine receptors
through amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. Upon binding
of cytokines to their receptors, Jaks are activated and phosphorylate the receptors,
creating docking sites for signaling molecules, especially members of the signal transducer
and activator of transcription (Stat) family. Mutations of the Drosophila Jak (Hopscotch) have revealed developmental defects, and constitutive activation of
Jaks in flies and humans is associated with leukemia-like syndromes. Through the generation
of Jak-deficient cell lines and gene-targeted mice, the essential, nonredundant functions
of Jaks in cytokine signaling have been established. Importantly, deficiency of Jak3
is the basis of human autosomal recessive severe combined immunodeficiency (SCID);
accordingly, a selective Jak3 inhibitor has been developed, forming a new class of
immunosuppressive drugs.

Gene organization and evolutionary history

Janus kinases (Jaks) are non-receptor tyrosine kinases and were discovered in searches
for novel protein tyrosine kinases using PCR-based strategies or low-stringency hybridization
[1-6]. In mammals, the family has four members, Jak1, Jak2, Jak3 and Tyrosine kinase 2
(Tyk2). In humans, the Jak1 gene is located on chromosome 1p31.3 and Jak2 is on 9p24; the Jak3 and Tyk2 genes are clustered together on chromosome 19p13.1 and 19p13.2, respectively. The
murine genes are located on chromosomes 4 (Jak1), 19 (Jak2) and 8 (Jak3 and Tyk2). Since the sequencing of other vertebrate genomes has been completed, we know that
there are four Jak family members in mammals, birds and fish (see the Additional data
files for alignments).

Jaks have been identified in the primitive chordate Ciona; it is unclear, however, whether this species only has a single Jak or whether more
will be found with further sequencing (see Additional data files). In Drosophila there is only one Jak kinase, Hopscotch (Hop) [7,8]. The ancestral Jak must therefore have arisen before the divergence of vertebrates
and invertebrates. Nematode worms and slime molds lack the family, however, but they
do express members of the signal transducer and activator of transcription (Stat)
family of transcription factors - which in vertebrates interact with Jaks, among other
proteins - suggesting that the Stats arose in evolution before the Jaks. It is of
interest that the expansion of the Jak kinases in higher animals occurred at the same
time as the evolution of innate and adaptive immune cells in fish; this is consistent
with the multiple roles of Jaks in immune cells (see below). Thus, cytokine receptors
acting via Jaks appear to have co-opted the Stat pathway for a variety of purposes,
especially for host defense. The proximity of the Jak3 and Tyk2 genes suggests that one may have arisen from the other by gene duplication, but it
is difficult to conclude which is the more ancestral. Jaks have approximately 20 exons;
alternatively spliced forms of Jaks have been described but their functional significance
is not known.

Characteristic structural features

The three-dimensional structure of the Jaks is at present unknown. This is no doubt
partly because they are relatively large proteins of more than 1,100 amino acids,
with apparent molecular masses of 120-140 kDa; expressing and purifying them has been
problematic. From the primary structure, putative domain structures have been recognized
that are conserved between mammalian, avian, teleost and insect Jaks. Seven Jak homology
(JH) domains have been identified, numbered from the carboxyl to the amino terminus
(Figure 1). The JH1 domain at the carboxyl terminus has all the features of a typical eukaryotic
tyrosine kinase domain. Interestingly, this domain is most closely related to the
kinase domains of the epidermal growth factor family of receptor tyrosine kinases,
suggesting that the Jak family may have arisen from this larger family of protein
kinases [9]. Adjacent to the JH1 domain is a catalytically inactive pseudokinase or kinase-like
domain (JH2), which is distantly related to other tyrosine kinase domains [9]. This tandem architecture of kinase domains is the hallmark of Jak kinases and gives
them their name; just like the Roman god Janus, they are two-faced with respect to
these domains. Although the pseudokinase domain lacks catalytic activity, it has an
essential regulatory function. A number of patient-derived and artificial mutations
within this domain abrogate kinase activity, underscoring its critical function [10,11]. Conversely, a mutation within this domain in Drosophila Hop activates the kinase and leads to transformation (discussed below) [12-14]. In mammalian Jak2, experimentally introduced mutations in this domain can also increase
basal activity, but they abrogate ligand-dependent activation [11,15].

Figure 1. A schematic representation of the primary structure of Janus kinases (Jaks), which
are made up of FERM, SH2-like, pseudokinase and kinase domains. An alternative nomenclature
for the putative domains is as a series of Janus homology (JH) domains. The FERM domain
mediates binding to cytokine receptors. Both the FERM and the pseudokinase domains
regulate catalytic activity and appear to interact with the kinase domain. Jaks autophosphorylate
at multiple sites (P), including two in the activation loop of the kinase domain,
but the precise function of these modifications is just beginning to be understood.

The amino terminus of Jaks contains an SH2-like domain (JH3-JH4) and a Band-4.1, ezrin,
radixin, moesin (FERM) homology domain (JH6-JH7). The FERM domain is 300 amino acids
long and is implicated in mediating interactions with transmembrane proteins such
as cytokine receptors; for some but not all cytokines, Jaks appear to be important
in regulating cell-surface expression of the cognate receptors [16,17]. In addition, the FERM domain binds the kinase domain and positively regulates catalytic
activity [18]. Unfortunately, the lack of crystal structures severely limits the understanding
of the intramolecular interactions that involve Jaks. Binding partners for the Jak
SH2 domain have not been identified.

Localization and function

In mammals Jak1, Jak2 and Tyk2 are ubiquitously expressed. In contrast, the expression
of Jak3 is more restricted; it is predominantly expressed in hematopoietic cells and
is highly regulated with cell development and activation [6,19,20]. At the cellular level, Jaks can be found in the cytosol when they are experimentally
expressed in the absence of cytokine receptors, but, because of their intimate association
with cytokine receptors, they ordinarily localize to endosomes and the plasma membrane,
along with their cognate receptors [21,22]. The link between Jaks and cytokine signaling was first made using mutant cell lines
that lacked responsiveness to interferon (IFN). One such cell line was shown to lack
Tyk2, and adding back this kinase restored IFN signaling [16]. Shortly thereafter other Jaks were shown to be associated with various cytokine
receptors [23-26], and subsequently Jak knockout mice have illustrated their essential and specific
functions (see Table 1).

A large number of cytokines are dependent upon Jak1, including a family that use a
shared receptor subunit called common γ chain (γc), which includes interleukin (IL)-2,
IL-4, IL-7, IL-9, IL-15 and IL-21. These cytokines are also dependent upon Jak3, because
Jak3 binds γc. Jak1 is also essential for another family that uses the shared receptor
subunit gp130 (IL-6, IL-11, oncostatin M, leukemia inhibitory factor (LIF), ciliary
neurotrophic factor (CNF)) as well as granulocyte colony-stimulating factor (G-CSF)
and IFNs. Jak2 is essential for the hormone-like cytokines such as growth hormone
(GH), prolactin (PRL), erythropoietin (EPO), thrombopoietin (TPO) and the family of
cytokines that signal through the IL-3 receptor (IL-3, IL-5 and granulocyte-macrophage
colony-stimulating factor, GM-CSF). Jak2 is also important for cytokines that use
the gp130 receptor and for some IFNs.

Tyk2 was the first Jak to be implicated in IFN signaling, but subsequent studies indicate
that Tyk2 is essential for IL-12 signaling but not for IFN-α/β signaling or for cytokines
that use gp130 [27,28]. Tyk2-/- mice also have defective responses to lipopolysaccharide (LPS, a component of the
outer membrane of Gram-negative bacteria), but whether this is a direct or indirect
effect has not been defined. In particular, a role for Tyk2 in signaling through the
Toll receptor, which mediates the response to LPS, has not been established [29,30].

Jak1 knockout mice have a perinatal lethal phenotype, probably related to the neurological
defects that prevent them from suckling [30] (Table 1). These mice also have defective lymphoid development and function as a result of
defective signaling by cytokines through Jak1. Jak2 deficiency results in embryonic
lethality at embryonic day 12.5 as a result of a failure in definitive erythropoiesis
[31,32]. Interestingly, Jak3 deficiency was first identified in humans with autosomal recessive
severe combined immunodeficiency (SCID) [33,34]. We now know that Jak3 binds to γc and that deficiency of either Jak3 or γc abrogates
signaling by the family of cytokines using this receptor subunit. Not surprisingly,
this has devastating consequences in terms of immune-cell development and function.
Together, mutations in the receptor for IL-7, γc and Jak3 account for two-thirds to
three-quarters of cases of SCID [35]. Jak3-/- mice were subsequently generated, and they too exhibit SCID but notably do not have
non-immune defects [36-38]; this is notable because it suggests that an inhibitor of Jak3 would have restricted
effects in vivo ([35]; see below).

The Jak/Stat pathway has been extensively studied in Drosophila and has been demonstrated to be involved in stem-cell maintenance, ovarian-cell migration
and sex determination [13,39]. In development, this pathway is important for embryonic segmentation and larval
hematopoiesis as well as for development of the eye, wing, trachea, hindgut and limb
[14,40-44]. A gain-of-function mutation in Hop has been identified that results in a leukemia-like phenotype in the affected flies;
this is designated tumorous lethal (Hoptum-l) [12,45,46]. In human leukemias, chromosomal translocations result in fusion proteins of the
Tel transcription factor with Jaks. This creates a constitutively active Jak, which
has also been documented to be transforming [47,48]. In human cells transformed with T-cell leukemia virus-1, Jak3 and Stat5 are constitutively
activated [49]. Constitutive activation of Stats is very common in many other types of tumors, although
the mechanisms underlying this activation have yet to be defined.

Jaks are constitutively associated with the membrane-proximal regions of cytokine
receptors, although in some cases interaction between the Jak and the receptor is
increased upon ligand binding (Figure 2). It has been proposed that ligand binding promotes a conformational change in the
receptor, which promotes Jak activation through reciprocal interaction of two juxtapositioned
Jak kinases and auto- and/or trans-phosphorylation of tyrosine residues on the activation
loop of the Jak kinase domain.

Figure 2. An overview of cytokine signaling. Cytokines bind to homodimeric or heterodimeric
receptors, which are constitutively bound to Jaks. Jaks are thought to be activated
by a conformational change in the receptor that allows trans- and/or auto-phosphorylation
of the two bound Jaks. These in turn phosphorylate the cytokine receptors. Stat proteins
bind the phosphorylated receptor chains, allowing the Jaks to phosphorylate the Stats.
Phosphorylated Stats form dimers and translocate and accumulate in the nucleus, where
they regulate gene expression.

Like other tyrosine kinases, Jaks undergo autophosphorylation, but the importance
of this modification in Jak-dependent signaling is not very well understood. Autophosphorylation
within the activation loop positively regulates kinase activity; in Jak3, however,
phosphorylation in this region can enhance or inhibit catalytic activity, depending
upon the site of phosphorylation [50] (Figure 1). Other sites of autophosphorylation have recently been identified. For instance,
a conserved residue in Jak2 and Jak3 that resides in the hinge region between JH1
and JH2 is a prominent site of autophosphorylation (Tyr813 in Jak2 and Tyr785 in Jak3)
[51]. This site serves to recruit the adapter protein SH2-Bβ, which positively regulates
Jak2 activity. Other sites of autophosphorylation in Jak2 include Tyr221 and Tyr570
[52].

Frontiers

Despite intensive studies during the past decade that have generated the model shown
in Figure 2, the exact molecular mechanisms of Jak activation have largely remained elusive.
It is clear that much more detailed structural information pertaining to Jaks and
the Jak-cytokine-receptor complex is needed to enhance our understanding of the mechanism
of Jak activation. Also, the exact mechanism and functional relevance of autophosphorylation
at different sites in Jaks is not known but will be an interesting area for future
research.

Another important topic for future studies is to define the mechanisms of crosstalk
between Jaks and other pathways. For instance, the receptor Notch has been reported
to promote Stat3 activation, and the Notch effectors Hes1 and Hes5 have been found
to associate directly with Jak2 and Stat3 [53]. Evidence for cooperation between the Jak/Stat and Notch pathways has also been provided
by work from Drosophila [54] and genetic screens in Drosophila have identified additional potential modifiers of the Jak/Stat pathway [55]. Jaks have also been reported to be activated by a variety of structurally diverse
receptors, beyond the cytokine receptors. Examples include receptor tyrosine kinases,
death receptors (such as CD40) and G-protein-coupled receptors (such as chemokine
receptors). Many of the studies have employed overexpression or putatively specific
inhibitors to implicate the Jaks, but we now know that these inhibitors are not specific,
so the essential function of Jaks for non-cytokine receptors remains uncertain. This
is clearly another critical area for future work.

Finally, because of the crucial role of Jak3 in cytokine signaling through γc and
because of its limited tissue expression, the inhibition of Jak3 activity has emerged
as a promising strategy for immunosuppression. A highly selective and potent Jak3
inhibitor (CP-69O, 550) has recently been developed that has nanomolar potency against
Jak3 in vitro, with much less potency against other Jak family members. Consequently, CP-690, 550
was both very efficacious and well-tolerated in animal models of organ transplantation
[56]. One might anticipate that this drug will help to overcome the unwarranted side effects
often seen in patients under current immunosuppressive therapy. Thus, the drug could
be useful in blocking transplant rejection and in the treatment of autoimmune diseases.
Conceivably, it might also be useful in treating those hematological malignancies
that exhibit constitutive Jak3 activation. Targeting Tyk2 with specific drugs would
also be logical, given its restricted role; presumably a Tyk2 inhibitor would be useful
in some immune-mediated diseases. Whether a Jak2 inhibitor would be useful in malignancies
is also worthy of consideration.

Additional data files

Protein sequence alignments in text and jpeg format are available for orthologs of
Jak1 (Additional data files 1 and 6), Jak2 (Additional data files 2 and 7), Jak3 (Additional data files 3 and 8), Tyk2 (Additional data files 4 and 9), and undefined members of the family (Additional data files 5 and 10), and a key for the alignments (Additional data file 11).